TWI237663B - Method of analyzing expression of gene - Google Patents

Abstract

A cDNA is prepared from mRNA. The prepared cDNA is subjected to incision with two different types of restriction enzymes. The sequence of a portion of each of the fragments obtained as a result of treatment is determined, and the fragments are classified on the basis of the determined sequence by using a primer set. At the same time, the fragments are amplified in a manner that the relative expression magnitude thereof continues to be reflected therein. When the fragments have been classified into a predetermined number of fractions, the respective fractions are subjected to electrophoresis, and a gene expression profile is produced from the results.

Description

1237663 A7 B7 V. Description of the invention (1) (Please read the notes on the back before filling this page) This application is based on Japanese Patent Application No. 2 0 〇〇— filed on February 12, 2012 No. 3 7 7 8 8 7 and claims priority based on the application. This application is incorporated into this specification by reference. (Technical Field) The present invention relates to a method for generating a gene expression analysis map and a method for analyzing a gene expression frequency. [Background Art] In 2000, the entire sequence of the human genome was continuously determined. The huge amount of data obtained from this is the basis for a comprehensive understanding of the networks that contain these gene products in all genes and specific cells. The methods used to explain such networks are as follows. For example, the Differential Display method disclosed in US5262311 and US5599672, and the sequential analysis method of genetic performance disclosed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs and the Ministry of Economic Affairs printed 1 0-5 1 1 0 0 2 ( That is, serial analysis of gene expression (hereinafter referred to as SAGE), and the microarrays and DNA fragments disclosed in USP 5807522, USP 5700637 and USP 5744305. The classification display method is a method using c D N A prepared by cells as a substrate. With respect to c D N A, various arbitrary gene expressions in a cell can be analyzed by performing PCR using plural types of anchor primers and arbitrary primers. However, this method is only a part of the analysis of all genes. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -4- 1237663 A7 B7 V. Description of the invention (2). In addition, the use of anchor primers and arbitrary primers has a problem of lack of reproducibility. (Please read the notes on the back before filling out this page.) In contrast, the method for obtaining a performance analysis map of all-expressing genes in a cell is SAG E. S A G E is analyzed using c D N A modulated by m R N A modulated by the cell. The adjusted c DNA was subjected to restriction enzyme treatment, a fragment of about 9 to 11 base pairs was cut out, and fragments from various c D N A were ligated, and then sequenced. However, in SAGE, in order to obtain data of about 50% of the full-expression genes, a sequence of about 100,000 cycles must be performed, which makes the cost very high. In addition, fragments from cDNA are short, and in fact, it is impossible to isolate genes from the fragments. The microarray of US 5 8 0 7 5 2 2 and the D N A fragments of USP 5700637 and USP 5744305 are used to immobilize probes of known genes in the solid phase. By hybridizing the sample with this probe, a method for analyzing the expression of genes can be obtained. In these methods, the sequence of the gene to be detected must be known. Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs [Disclosure of the Invention] The first object of the present invention is to provide a method for generating a performance analysis map of a gene in a wide range. A second object of the present invention is to provide a method for analyzing the expression frequency of a wide range of genes. The above-mentioned first purpose is to create a method for analyzing the expression of genetic expressions, which is to have the paper size applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) -5- 1237663 A7 B7 V. Description of the invention (3) ( a) For the process of m RNA extracted from cells to synthesize c DNA of its 3 'terminal addition target substance, (b) the process of cutting the obtained reaction product with the first restriction enzyme X, (c) the process of The X aptamer having a complementary sequence to the sequence of the restriction enzyme cutting site of the first restriction enzyme X is used to combine the fragments obtained in the engineering b, (d) by binding a substance having a high affinity to the target substance 'Then the process of recovering the fragment obtained from project c can be recovered. (E) The fragment recovered from project d can be cut with the second restriction enzyme Y, and the fragment containing the cleaved c DNA can be obtained. 5 The project of 5 side fragments, (f) For the fragment obtained from project e, a project in which a Y aptamer having a complementary sequence to the sequence of the second restriction enzyme Y recognition cut site is added, (g) using the X adaptor sequences have A primer with a complementary sequence and its 3 'terminal with any two base sequence NN, and a primer with a complementary sequence to the Y adaptor sequence and its 3' terminal with any two base sequence NN, and for the aforementioned project f. The obtained fragment is subjected to a PCR reaction project. (h) The obtained PCR product is subjected to electrophoresis, and a method of preparing a gene expression analysis map can be achieved by detecting a moving distance and a wave peak. The above second purpose is to analyze the frequency of gene expression, which is based on the Chinese paper standard (CNS) A4 specification (210 × 297 mm). (Please read the precautions on the back before filling this page) Order the wisdom of the Ministry of Economic Affairs Printed by the Consumer Cooperative of the Property Bureau • 6-1237663 A7 B7 V. Description of the invention (4) Yes (Please read the precautions on the back before filling this page) (a) For both control cells and test cells, use the above production The method of gene expression analysis chart, the process of making the gene expression analysis chart, (b) the method of analyzing the change of the gene expression frequency of the tested cells can be achieved by comparing the two gene expression analysis charts obtained by comparison. Still other objects and advantages of the present invention are described in the following description and examples. Based on these records and examples, the present invention can be understood more clearly. In addition, the objects and advantages of the present invention can be explained in detail and achieved through the methods and combination means shown below. (Brief description of the drawings) The drawings incorporated in this specification and forming a part of it are necessarily detailed descriptions of the preferred aspects of the present invention, as well as the above general descriptions and preferred aspects described later. It is to explain the basic idea of the present invention for use. Fig. 1 is a schematic diagram showing a method of making a gene expression analysis map according to an aspect of the present invention. Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs Fig. 2 is a flow chart showing a method of making a genetic expression analysis map according to an aspect of the present invention. Fig. 3 is a diagram showing an example of a part of a gene expression analysis map obtained according to an aspect of the present invention. FIG. 4 is a combination diagram showing arbitrary two base sequences. Fig. 5 is a diagram showing the proportion of genes detected in the analysis map of gene expression according to aspects of the present invention. This paper size applies the Chinese National Standard (CNS) A4 specification (21.0X297 mm) 1237663 A7 B7 V. Description of the invention (5) Figure 6 is a diagram showing a preferred example of the X adaptor and the Y adaptor. (Please read the precautions on the back before filling this page.) Figure 7 shows a partial gene sequence diagram of human arylamine N-acetamidine transferase obtained from the database. FIG. 8 is a diagram showing an example of data about fragments obtained by restriction enzymes. Fig. 9 is a partial diagram showing an example of a gene expression analysis diagram showing the expression of P 2 1. Fig. 10 is a partial diagram of an example of a gene expression analysis diagram showing m d m 2 expression. Fig. 11 is a partial diagram showing an example of a gene expression analysis diagram showing cyclic G expression. FIG. 12 is a diagram showing the composition of the m R N A modulator used in Example 2. FIG. FIG. 13 is a partial diagram showing a gene expression analysis map obtained in Example 2. FIG. Fig. 14 is a partial diagram showing a gene expression analysis map obtained in Example 2. Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs [The best form for implementing inventions] 1 · What is the invention? The inventors and others have discovered that the classification of genes expressed in specific cells can be greatly changed. The complexity and cost-effectiveness of operations in making gene expression analysis maps. On the basis of this finding, we made intensive research, and the result reached the invention. According to one aspect of the present invention, in order to provide a one-time and simple method for producing paper, the Chinese national standard (CNS) A4 specification (210X 297 mm) is applicable. -8-1237663 A7 B7 5. Description of the invention (6) (please first (Read the notes on the back, and then fill out this page.) This is a method for analyzing the expression of a wide range of genes including approximately all genes expressed in specific cells. By recognizing approximately all the genes expressed, more genes can be identified than before. One aspect of the present invention is a genetic expression analysis map method developed based on the restriction enzyme D N A fragment length and the polymerase chain reaction (ie, PCR). Through such a gene expression analysis map method ', almost all the genes represented can be identified, that is, both known genes and unknown genes can be identified as well. In addition, this method does not miss a single gene 'and can be identified and detected separately. In addition, the frequency of its performance can also be determined. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. One of the basic features of the method for producing the gene expression analysis map of the present invention is to classify genes expressed in specific cells as follows. The number of mRNAs expressed in a particular cell is considered to be about 20,000. First, c DNA is synthesized from the expressed mRNA. The obtained double-stranded c DNA was cut with appropriate two restriction enzymes to make various fragments having only recognizable lengths. Thereafter, it was classified into 256 fractions which could identify a partial sequence of the obtained fragment. This classification was first performed using 2 5 6 primer sets of arbitrary design. Under the condition that the relative performance is still reflected, the fragment is enlarged in each primer group and the fragment is classified. For classification, the obtained fractions, for example, each of 2 to 6 fractions, are subjected to electrophoresis per fraction to separate the components. In this way, the performance gene data obtained from one cell is classified to the extent that it can be parsed. In this way, it is possible to easily produce almost all genes without missing them, and accurately capture a gene expression analysis map of each gene expression. For example, use Figure 1 to illustrate the specificity of the manual paper grades classified into 2 56 fractions. The national standard (CNS) A4 specification (21〇 × 297 mm) -9-1237663 A7 ___B7 V. Description of the invention ( 7) (Please read the notes on the back before filling out this page) Printed section of the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. ^ D A A group 2 was synthesized from the expressed m R N A group 1. By cutting it with two appropriate restriction enzymes, the c D n A fragment group 3 can be obtained. According to the sequence of 2 test bases and 4 bases of each terminal of c D. NA, that is, according to these sequences, adenine (hereinafter referred to as A) and guanine (hereinafter referred to as G). Either cytosine (hereinafter referred to as c) or thymine (hereinafter referred to as T) is classified. That is, according to 5, the bases of the terminal (shown in black in FIG. 1) are first classified into 4 groups of 4. It is further classified into 16 groups 5 according to the next test base, and then 3, the second test base of the terminal is classified into 6 4 groups 6, and the base of the terminal is further classified into 2 5 6 according to 3. Group 7 If it is inferred from the general expression mRNA type, the 256 groups 7 finally obtained contain about 80 to about 100 kinds of c D N A, respectively. That is, if each group is subjected to electrophoresis, about 80 to about 1000 peaks can be detected. Therefore, the total m R N A obtained from a specific cell is represented by 25 graph patterns having about 80 to about 100 peaks. That is, from these 256 graphs, an analysis graph of the expressed genes is constructed. For example, an example of a chart contained in such an analysis diagram is shown in Figure 3. The graph of Fig. 3 is a graph showing the components contained in one fraction obtained by fractionating the fractions, magnifying the fractions by PCR, and electrophoresing the reaction product. In addition, one of the characteristics of the present invention is that c D ΝA obtained by expressing m R N A is appropriately cut using two appropriate restriction enzymes, preferably M s ρ Ϊ and M s e I. By appropriate cutting, the above classification can be achieved. Hereinafter, the present invention will be described in more detail. This paper scale applies Chinese National Standard (CNS) A4 specification (210X297 mm) -10- 1237663 A7 B7 V. Description of the invention (8) 2 Detailed description (1) Gene expression analysis chart The method for making the gene expression analysis chart of the present invention is basically The above is equipped with the following works. That is, (a) a process for synthesizing c DNA of a 3-terminal addition target substance from m RNA extracted from a cell, (b) a process for cutting the obtained reaction product with a first restriction enzyme x, (c ) An engineering process in which an X aptamer having a complementary sequence to the restriction enzyme cutting site of the first restriction enzyme X is combined with the fragment obtained in the engineering b, (d) has a high affinity for binding to the target substance Substances' can be recovered in the process of the fragment obtained from the project c, (e) the fragment recovered in the process d is cut with a second restriction enzyme Y, and the fragment containing the cut substance c can be obtained by removing the fragment bound to the target substance Project of DNA 5 'side fragment, (f) For the fragment obtained from project e', a project in which a Y aptamer having a complementary sequence to the sequence of the second restriction enzyme Y recognition cut site is added, (g) is used A primer having a complementary sequence to the X aptamer sequence and its 3 'terminal having an arbitrary two base sequence NN' and a complementary sequence to the γ adaptor sequence and an arbitrary two base sequence to its 3 'terminal NN Primer and for The project for the PCR reaction of the fragment obtained from the previous project, this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, 1T-1237663 A7 B7 V. Description of the invention (9) (h) The electrophoresis of the obtained p CR product, through the detection of the moving distance and the wave peak, the analysis of the gene expression is made. engineering. Here, although not particularly limited, the 5 'side of the sense strand (the same sequence as the mRNA of the template) is regarded as the 5' side of the double-stranded c DNA, and the 3 'side of the sense strand is regarded as the double-stranded c 3, side of DNA. A specific example of a method for generating a gene expression analysis map of the present invention will be described with reference to FIG. 2. Here, each letter in FIG. 2 represents the base constituting the base sequence ° A represents adenine (hereinafter abbreviated as a), and g represents guanine (

Hereinafter referred to as G), C is referred to as cytosine (hereinafter referred to as G) and T is referred to as thymine (hereinafter referred to as T). Here, N, W, X, Y, and Z represent arbitrary bases. X and γ and w and Z are mutually complementary. In addition, the above-mentioned projects (a) to (h) and the projects 3 to h in FIG. 2 correspond to each letter, respectively. First, m R N A 1 1 ο was extracted from a specific cell as a test object. The extracted m R N A 1 1 3, the terminal poly A tail complementary oligo-d T primers, were labeled with biotin 1 3. This was used as a primer to synthesize c D N A to obtain double strands 1 2 (Figure 2, Project a). Here, an example using biotin as a target substance is shown. The double-stranded 12 was cut using the four-base recognition restriction enzyme μ s p I as the first restriction enzyme X (FIG. 2, project b). Here, an example using M s p I as the first restriction enzyme X is shown. Thereafter, streptavidin 14 was used to capture biotin 13. This captures the 3, side of the cut double strand c D Ν Α (Figure 2, the paper size of this project applies the Chinese National Standard (CNS) A4 specification (210 × 297 mm)) (Please read the precautions on the back before (Fill in this page)

Printed by 1T Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs -12-1266366 A7 B7 V. Description of Invention (10) c). Here, examples of substances having high affinity using streptavidin as a target substance are shown. On the 5, side of the recovered double-stranded c D N A, an X adaptor 15 having a complementary sequence to the recognition restriction site of the first restriction enzyme X, that is, M s p I (FIG. 2, project d) is bound. It was cut using the restriction enzyme M s e I of the second restriction enzyme Y (FIG. 2, process e). Here, an example using M s e I as the second restriction enzyme Y is shown. Secondly, a Y aptamer 16 having a complementary sequence to the restriction enzyme Y, that is, the recognition cut-off site of M s e I was added (Fig. 2, project f). After the above processing, a double-stranded sequence 17 containing a known sequence can be constructed on the double-stranded two-terminal terminal. Next, use this double-stranded sequence 17 as a template, and use the 5 ′ side (for antisense strand) PCR primers 18 of the double-stranded DNA labeled with fluorescein and the double-stranded c without fluorescent marker. The 3, side (for sense strand) primer 19 of the DNA was subjected to a PCR reaction (Fig. 2, engineering g). Here, the X primers 18 and Y primers 19 for the P CR are complementary sequences to the sequences of the aforementioned aptamers, that is, the X and Y adaptors, respectively, and have 2 bases in the direction of amplification. Base sequence. The arbitrary two bases mentioned above are designed to contain all sequences obtained by combining four bases of A, G, C, and T, so that a total of 2 56 types of primer sets can be obtained. That is, using these primer sets, performing P CR ′ on all the double-stranded c D N A can classify all the existing c DNA into 25 groups and perform PCR amplification at the same time. Any four-base combination of this primer set indicates that the paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) f Please read the notes on the back before filling this page; >-Order the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by Employee Consumer Cooperatives-13-1237663 A7 B7 V. Description of the invention (10 is shown in Figure 4. The combination of AA — AA to TA — GA is recorded in Figure 4 (please read the precautions on the back before filling this page) In the final project of Figure 1, Project h, the obtained PCR products of 256 fractions were subjected to electrophoresis, and the peaks were measured to obtain an analytical map of gene performance (Figure 1, Project h). One of the obtained 256 fractions The results obtained when the fractions are subjected to electrophoresis are obtained, for example, in the graph shown in Fig. 3. The vertical axis shows the expression amount using the fluorescence intensity as an index. The horizontal axis shows the molecular weight using the moving distance of the electrophoresis as an index. In addition, the order of the aforementioned project c and project d may be changed. That is, the aforementioned project d may also be performed before the project c. Furthermore, the first restriction enzyme is used as the second restriction enzyme, and the second restriction enzyme is used. As the first restriction enzyme, More genes can be cut off, which can improve the detection sensitivity. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, the double strands 12 obtained from the above project a are divided into two groups, c DNA mixture A and c For DNA mixture B, for c DNA mixture A, the processes b to h are performed as described above, and in parallel or sequentially, c DNA mixture B can also be processed as follows. That is, c DNA mixture B is made using the restriction enzyme M se I Is the first restriction enzyme, and the restriction enzyme M sp I is used as the second restriction enzyme, and the other points are treated in the same manner as above. In this way, by using the first restriction enzyme and the second restriction enzyme interchangeably, Genes that cannot be detected can also be detected. Specifically, the treatment of c DNA mixture B is to cleave the double strands 12 contained in c DNA mixture B using a four-base recognition restriction enzyme M se I. Combined with the identification of the restriction enzyme M se I, the cut size of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) '' " — -14- 1237663 Α7 Β7 5. Description of the invention (d (please first Read back (Notes on this page, please fill out this page again)) M se I aptamers with complementary sequences, use streptavidin to capture biotin, and recover the 3 'side of the double stranded strand 12. Second, limit its use The enzyme M sp I is cut off. Then, an M s ρ I aptamer with a complementary sequence is added to the recognition cut site of the restriction enzyme M s ρ I. After the above processing, the double stranded 12 2 is constructed. The terminal contains a sequence of a known sequence. Second, for this sequence, a PCR reaction was performed on the cDNA using X primer 18 marked with a fluorescent pigment and 19 primers without a fluorescent marker. Here, the aforementioned two primers 18 and 19 are substances in which the aptamer, that is, the Y aptamer and the X aptamer have complementary sequences, and are added with two bases in the amplification direction. The additional two bases are designed to contain the entire sequence of four base combinations of A, G, C, and T, so a total of 2 5 6 primer sets can be obtained (Figure 2 project a to project f The specific 2 5 6 kinds of NNs — the printed NN test sequence of the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs are shown in Figure 4). That is, by using these primer sets to perform P C R on all c D N A, all types of c DNA existing can be classified into 2 5 6 groups. The obtained 256-group PCR products were subjected to electrophoresis, and the moving distances and wave peaks were measured to obtain a gene expression analysis map. The expression genes classified according to the method of the present invention are as shown in FIG. 5, for example, in the case of mice, about 85% of the genes of 100 rats are randomly selected for identification and detection. That is, in the case where M s ρ I is used as the first restriction enzyme and M s e I is used as the second restriction enzyme, about 66% of the expressed genes are cut off. In the case where M se I is used as the first restriction enzyme and M s ρ I is used as the second restriction enzyme, the approximate paper size of the expressed gene applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -15- 1237663 A7 B7 V. Description of the invention (13) (Please read the precautions on the back before filling in this page) 19% is cut off. Therefore, by exchanging the first restriction enzyme and the second restriction enzyme, about 85% of the expressed genes as a whole are detected and detected. Thereby, a gene analysis map can be made more accurately. The percentage of genes identified by previous methods is usually 20 to 30%, with a maximum of 50%. Therefore, the proportion of genes identified in the analysis map of gene expression made according to the aspect of the present invention is much higher than the previous proportion. Theoretically, it can be said that the genes contained in 1 cell can be almost completely identified. The term "analysis map of gene expression" as used herein refers to the gene expression pattern in a specific cell under certain conditions, and includes information on the presence or absence of known and unknown gene expression, and the expression amount of all genes expressed. The gene expression analysis map can be used as a means for analyzing gene expression. The "poly A tail" as used herein refers to the 3 'terminal sequence of m R N A, also commonly referred to as poly A. In addition, cDNA can be synthesized from an mRNA having the polyA tail via an "oligod T primer" having a complementary sequence to the polyA tail. The "oligo dT primer" used herein is also commonly referred to as an oligo (dT) primer. Synthesis of cDNA with oligo-dT primers can be achieved under any conditions generally performed. The "subject matter" and "substances with high affinity to the target substance" printed here by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs refer to those who have high affinity with each other and constitute a specific combination. substance. The above item, that is, in the example described in "(1) Gene expression analysis map", although biotin is used as the target substance and streptavidin is used as the target substance, the substance has high affinity, But it is not limited to this. They can be used as long as they have a high affinity for each other and can bind specifically. The target substance that can be used in the present invention and the standard paper size are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) '~~ -16-1237663 A7 _____ B7__ V. Description of the invention (M) (Please read the back first (Please fill in this page before filling in this page) Examples of substance combinations with high affinity include biotin and streptavidin, biotin and avidin, FI TC and FI TC antibodies, DIG and anti-DIG, proteins A and rat I gG, and latex particles are not limited thereto. In each combination, any one can be used as a target substance, and any one can be used as a substance having a high affinity for the target substance. Printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The "restriction enzymes" used here are generally enzymes that are also known as restriction kernel enzymes. They are enzymes that hydrolyze and cut off double-stranded DNA in a specific sequence. In the method according to the aspect of the present invention, two restriction enzymes X and Y are used in combination in order to obtain a suitable fragment. The restriction enzyme that can be used in the present invention is preferably an enzyme that can cleave a double strand composed of c D N A synthesized by m R N A expressing a gene into a fragment having a recognizable length. In addition, the obtained double strand is cut into more, preferably an enzyme that cuts almost the complete double strand. Examples of such enzymes are shown in Table 1. Any two enzymes are selected from the enzymes in Table 1, and they can be used in combination. The enzymes shown in Table 1 are four base recognition enzymes, but other four base recognition enzymes or six base recognition enzymes may be used. In particular, in the method according to the aspect of the present invention, it is preferable to use a tetrabasic recognition enzyme, and more preferably to use M s p I and M s e I in combination. In the above example, M s p I (or M s e I) is used as the restriction enzyme X, and MseI (or MspI) is used as the restriction enzyme Y. This paper size applies to China National Standard (CNS) A4 (210X 297 mm) -17- 1237663 A7 B7 V. Description of Invention (15) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs

Table 1 Acc II c0yco Hpa II C / CGG AT8I QT / AC Ηδρ92 II CATO / Alul AG / CT HtpAI G / CGC AspLEI 6CG / C kzofil / GATC Bfal C / TAG Mae IC / TAG BscFI / GATC Mbol / GATC Beh12361 CGJCG Msel T / TAA Bshl GQ / CC Msp I (^ CGG BsiS! C / CGG Mvnl CG / CG Ββρ143I / GATC Nde II / GATC BstU I CQ / CQ Nla III CATO / BsuFU GQ / CC Pall QO ^ CC C Cut I OOQIC Rsal GT / AC CspBI G / TAC Seu3A 1 / QATb Wpn II Mat. 幺 se91 / AATT FnuD II co / co Taql T7CQA Hae III 6G / CC Thai C0 / CQ Hap II C / C ^ G Trull ΤΑΓΑΑ Hhal GCG / C TruSI T / TAA Hin2l C / CGO Tip5091 / AATT Hin6 Book G / CGC TspE 1 / AATT HinP11 O / CGC TthHBB 1 T / CQA (Please read the precautions on the back before filling this page) This paper standard is applicable to Chinese National Standard (CNS) A4 specifications (210X 297mm) -18-1237663 Α7 Β7 V. Description of the invention (16) (Please read the precautions on the back before filling this page) The "adaptor" used here is used to enlarge the final pc R The primers used at the time are bound. The adapter used is designed based on the restriction enzyme used. That is, the binding limit The X aptamer of the recognition cut site of enzyme X contains a complementary sequence to the recognition cut site of restriction enzyme X, and may also have an arbitrary sequence. Furthermore, the efficiency of PCR can be considered for any sequence and the length of the base. And the design. It is better to design the X adaptor to about 15 bases. In this way, stable pc R can be carried out. The Y adaptor combined with the restriction enzyme Y recognition cutting site contains the recognition enzyme for restriction enzyme Y. The broken site is complementary, and it can have any sequence again. The arbitrary sequence and the length of the base can be designed considering the efficiency of PCR, etc. It is better to design the Y adaptor to about 15 bases. Stable PCR can be performed. For example, the preferred X adaptor sequence when M sp I is used as the restriction enzyme X is shown in FIG. 6 (a), and when M se I is used as the restriction enzyme X,

The preferred X adaptor sequence is shown in Figure 6 (b). In addition, a better adaptor sequence when M sp I is used as a restriction enzyme is shown in Figure 6 (c). The best aptamer sequence is shown in Figure 6 (d). However, it is not limited to this. The "primer set" used in project g is composed of a set of X and Y primers used by the double-stranded c D N A obtained in project f by P C R amplification. Specifically, as described above, the "arbitrary two-base sequence N N" used herein is a sequence arbitrarily selected from adenine, thymine, guanine, and cytosine. As mentioned above, when an arbitrary sequence is used as the two bases (that is, 'NN), the graph obtained by PCR of 1 sample contains about 80 to about this paper size. The Chinese National Standard (CNS) A4 specification (21〇) > < 297 mm) -19- 1237663 A7 B7 V. Description of the invention (j (please read the notes on the back before filling in this page) 100 peaks. Here, two of each sequence are used as two The base is the result of considering the simplicity and accuracy of the method. Therefore, in the method according to the aspect of the present invention, the preferred arbitrary sequence is a two-base NN, and the preferred number of primer sets is 2 5 6. It is not limited to this. The arbitrary two-base sequence NN contained in the two primers can also be made into three bases for any primer (X or Y primer) or both primers (X and Y primer). The above can increase the types of primers contained in the primer set. At this time, the obtained fractions are 1024 or 4096 respectively. In addition, according to the aspect of the present invention, in order to make the detection after PCR easier, it is better than Any terminal of the primer is bound to a fluorescent substance in advance. The 5 'terminal of the X primer having a complementary sequence to the X aptamer. The fluorescent substance that can be used in the method of the present invention includes 6-carboxyfluorescein (hereinafter, referred to as FAM), 4, 7, 2, 2, 4, , 5, 7, 7, hexachloro-6-carboxyfluorescein (hereinafter, referred to as HEX), NED (Applied Biosystems Japan) and 6-carboxy-X-rhodamine (hereinafter, referred to as R OX) Etc. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs: The P c R reaction implemented according to aspects of the present invention can be performed under normal conditions. For example, at 9 5 t: 1 minute, (9 5 ° C 20 seconds, 6 8 ° C 30 seconds, 7 2 ° C 1 minute) X 2 8 cycles, 60 ° C 30 minutes, etc. The electrophoresis means that can be used according to aspects of the present invention, if It can be used by electrophoresis means that can generally separate samples based on molecular weight. For example, Sequencar ABI PRISM 3 10 0 (Applied Biosystems J apa η) and Mega BACE 1 0 0 0 (This paper size applies to Chinese national standards (CNS) A4 specification (210 X tear mm) -20-1237663 A7 B7 V. Invention (18)

Amersham Pharmacia). (3) Identification of peaks (please read the precautions on the back before filling this page). Furthermore, according to the aspect of the present invention, the genes contained in each peak of the graph obtained as described above can also be identified. By identifying this peak, it is possible to identify genes that are expressed in a specific situation, or that the amount of expression is increased or decreased. The gene can be identified by recovering the peaks in the graph, for example, by experimental methods such as determining the sequence in general, and determining the sequence can be identified. In addition, without using the experimental method described above, it can also be obtained theoretically on a computer. For example, the data obtained from commonly available databases are identified on a computer based on the molecular weight of the restriction enzyme recognition site used and the fragments obtained. Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs. The length of the fragment obtained when any gene sequence obtained by the database is cut with a specific restriction enzyme, and the identification site of the restriction enzyme can be easily determined on the computer display. On the other hand, in fact, the length of a fragment obtained by restriction enzyme cleaving used in this method can be elucidated by performing electrophoresis. Therefore, if the adaptor sequence used is considered, the gene from which the fragment is derived can be identified without experimental analysis. An example of a theoretical identification method using a computer is described in Example 1 described later. The computer that can be used here may be a computer that is generally used. For example, a device including an input unit such as a keyboard and a mouse, an output unit such as a printer and a display, and an arithmetic unit such as a CPU can be used. In addition, examples of databases that can be obtained include GenBank, and this paper size applies the Chinese National Standard (cns) A4 specification (21 ox297 mm) ~ '-21-1237663 A7 B7 V. Description of the invention (19) EMB L and DDBI, etc. The data of other DataBanks, such as the public DataBank and commercial databases, is not limited to this. (Please read the notes on the back before filling out this page.) You can also combine experimental and theoretical methods. (4) Analysis of gene expression frequency In the method for generating a gene expression analysis chart according to the aspect of the present invention, the gene expression amount expressed by the test cell reflects the size of each peak shown in the chart. Therefore, by observing the change in the peak size, the expression frequency of the gene can be analyzed. For example, a comparison between normal cells and abnormal cells, a comparison between normal cells and cancer cells, a comparison between different cells, and a comparison between cells treated under different conditions can be performed. In addition, if a gene expressed or changed in a specific stimulus is identified according to this method in advance, a part of the gene expression analysis map can be created in subsequent experiments using only primers related to the specific gene. In this way, the expression frequency of the target gene can be analyzed. Printed in Example 1 by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The effects of radiation on P 2 1, mdm 2 and Cyc 1 in G performance are as follows. Review the radiation on P 2 1, mdm 2 and cyclin G performance. Influence. The mR N A obtained after irradiation of the rat breast cancer cell line S R-1 with radiation and the m R N A obtained from the unirradiated cells were used to make a gene expression analysis map and compared. This paper scale applies Chinese National Standard (CNS) A4 specification (21〇'297 mm) -22-1237663 A7 B7 V. Description of the invention (20) 1 · Genetic analysis map production Actual production According to the aspect of the present invention Analysis of gene expression. Koichi-1 mRNA extraction and cDNA synthesis Murine breast cancer cell line S R-1 (derived by Dr. Oyama, Yokohama City) was cultured in an αMEM culture medium in a 7 cm 3 flask (Falcon). This cell was irradiated with radiation from the upper side using 7 G y of P A N T A C Gai Jin Co., Ltd. The irradiation time was 3 hours. In addition, unirradiated cells were prepared in parallel as a control group. From each cell, 20 micrograms of total mRNA was extracted using Fast Track 2.0 Kit (Invitrogen). 20 micrograms of the extracted mRNA was mixed with 0.8 microliters of 5′-biotinylated dT primer (BRL) of 100 pmole and incubated at 65 ° C for 5 minutes. After ice-cooling, it was cultured in 20.0 microliters of reverse transcription buffer with a final concentration of 5 mM MgCl2 and 0.5 mM dNTP mixture (BRL) and 10 mM DTT (BRL) in culture 60. minute. Then 'put it in 150 μl of double-stranded synthetic buffer, with a final concentration of dNTP mixture (BRL) of 0. 27 mM and DTT (BRL) of 1.33 mM and 20.0 units E. coli ligase (BRL), 40.0 units of E. Coli DNA polymerase (Brl), and 2.0 units of RNaseH (BRL) were incubated at 16 ° C for 120 minutes, and thereafter at 7 ° C. Incubate for 15 minutes to stop the reaction. Use the paper size obtained by applying the Chinese National Standard (CNS) A4 (21〇 '乂 297 mm) (Please read the precautions on the back before filling this page).

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs of the Ministry of Economic Affairs of the People's Republic of China. 1237663 A7 B7 V. Description of the invention (21) The reaction product is divided into two equal parts and treated as reaction product mixture A and reaction product mixture B. 1 to 2 * Treatment of reaction product mixture A The reaction product mixture A is treated as follows. Here, M s p I is used as the first restriction enzyme, and M s e I is used as the second restriction enzyme. First, in 100 microliters, a final concentration of 20 units of the restriction enzyme M s p I (Takara) and 10 micrograms of the reaction product mixture A containing mRNA were reacted at 37 ° C for 360 minutes. After the reaction, it was refined with ethanol (500 µl x 3 times). Next, it was used in 15 microliters of T4 DNA ligase buffer using 5.0 micrograms of the X aptamer (BRL) with the GC sequence (that is, the complementary sequence of the fragment break site of the restriction enzyme M sp I). Company), and 10 units of T4 DNA ligase (NEB). Thereafter, magnetic beads with immobilized streptavidin (Dinal) were added to the reaction solution. For the immobilized streptavidin, the biotin contained in the double strands in the reaction solution is combined to obtain a linked product. Thereafter, it was reacted in 200 μl with a restriction enzyme Ms el (NEB) at a final concentration of 50 units at 37 ° C for 360 minutes. Transfer the supernatant to another tube and refine with ethanol (1,000 μl x 3 times). Thereafter, it was incorporated in 10 μl of T4 DNA ligase buffer with a 10 pmole Y aptamer having the AT sequence (that is, the complementary sequence of the fragment break site of the restriction enzyme M se I) (BRL) Use 10 units of T 4 DNA ligase (This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back first and fill out this ΐ tr Printed-24- 1237663 Μ __Β7__ V. Description of invention (d NEB) for ligation. (Please read the notes on the back before filling this page) Second, perform PCR on the resulting ligation product. Here, the χ aptamer has The 5 'side of the X adaptor of the complementary sequence combines any of the three fluorochromes FAM, NED, and NED. Furthermore, the X adaptor has an arbitrary two-base NN at the 3' side. On the other hand, it is at Y The Y aptamer with complementary sequences of the aptamer is an arbitrary two-base NN on the 3 'side. The combination of each NN and the fluorescent pigment is shown in Figure 4. In Figure 4, the substances used in the flag are , With the sequence marked by FA Μ at Column a is the same, EX is described in column b, NED is described in column c, and any two base sequence is described in the form (NN of X primers)-(NN of Y primers). Here, although Three fluorescent probes are used, but they are used to improve work efficiency. Therefore, even a single fluorescent probe can be used for the same purpose. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The reaction solution was diluted to 6 1 2 microliters using Tris-HCl buffer (hereinafter referred to as TE). For this purpose, 1 microliter of the diluted reaction solution was used. One sequence, that is, mixing 1 microliters of each solution containing a) FA MH "AA-AA", b) HEX "CT-AA" and c) NED "CA-AA" primers. Similarly, the primers of the sequence described in each position were mixed with 1 microliter of the diluted reaction solution in three groups. a) Since there is no Η E X and N E D up to the 8th to the 9th of the FAM, they are separately mixed with 1 microliter of the diluted reaction solution. The P C R reaction products produced by the 256 primer sets according to the above operation were used as 96 samples for electrophoresis. This standard is applicable to the Chinese National Standard (〇 奶) 8.4 (210 father 297 mm) 1 -25-1237663 A7 B7 V. Description of the invention (Z3) i -------- »clothing! (Please read the precautions on the back before filling out this page} Electrophoresis was performed on these 9 6 samples for electrophoresis. The electrophoresis was performed using a Maotian tube sequencer (ABI PRISM 3100, APpiied Bio systems Japan), and the swimming voltage was 1 5 Performed under the conditions of k V and swimming time of 2000 seconds. The electrophoresis results are obtained on the horizontal axis with a molecular weight using a moving distance as an index, and on the vertical axis with a fluorescence intensity as a graph indicating a lot of money. Although the sample contains P c R products marked by three fluorescent substances, they can be discriminated by changing the wavelength. 1-3 The reaction product mixture B is processed except that M se I is used as the first restriction enzyme and M sp Except for I as the second restriction enzyme, the reaction product mixture B was treated in the same manner as in the reaction product mixture A. In addition, in the same manner as the reaction product mixture A, 9 6 samples were subjected to electrophoresis to obtain a graph. 1-2 In the same way, use the electrophoresis result chart to obtain the gene expression analysis chart. 1-4 Analysis of the gene database. Use the data in the database to identify the genes contained in the peaks detected in the analysis chart, and obtain information about the restriction enzyme cut-off site used and the fragments obtained through the cut-off. First, the mRNA of the GenBank database is clarified. The full-length genes and all the data obtained from the EST registered only a part of the sequences are aggregated, and each type of data from each gene is organized into one. The common sequence is obtained from the aggregated data. The common sequence and a part used to constitute the common sequence This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) -26- 1237663 A7 B7 V. Description of the invention (The y-sequence is shown in Figure 7 (Figure 7). The sequence shown at the top is a common sequence. The "common sequence" as used herein refers to a common sequence of the same part of a gene, which is obtained by determining a high-probability common sequence among all sequences identifying a plurality of sequences. Figure 6 shows the human arylamine N-acetamidine Part of the gene sequence of the transferase. Secondly, for this common sequence and all the data used to form the common sequence, The recognition sequence of restriction enzyme X closest to the 3 'side was detected. Second, the recognition site of restriction enzyme X was detected starting from the recognition sequence of restriction enzyme Y closest to the 3' direction. The recognition sequence of M sp I was C / CGG, It was cut at the position "/". Furthermore, the recognition sequence of M se I was T / TAA. Furthermore, the fragments of the restriction enzymes X and Y obtained in this way were cut, and the number of bases of D NA was theoretically calculated. An example of the data obtained as described above is shown in Figure 8 (Figure 8). It can be seen from Figure 8 that the 104 bp slice break is obtained from the data of the GenBank database and many of the ES registration data (refer to Figure 8 " Length "column. However, it is also suggested from this data that there are mutations or sequence read differences in several data, and it is also possible to obtain 2 3 b p slice breaks (refer to the "length" column in Fig. 8). Next, from this gene database, the genes that showed increased radiation exposure, p 2 1, mdm2, cyclin G, and gadd 4 5 were investigated for their cut lengths and the two base sequences inside the restriction enzyme recognition site. 1 — 5 Identification of genes This paper applies Chinese National Standard (CNS) A4 specifications (210 X297 mm) (please read the notes on the back before filling out this page) -27- 1237663 A7 _____B7 V. Description of the invention (25) (Please read the notes on the back before filling out this page) After a comparative review, review the data obtained from the above 1-2 and 1-13, and the data obtained from 1-4. Identification of genes contained in the peaks detected by the gene expression analysis map of the present invention can be performed. Based on the cut length of p 2 1, mdm 2, cyclin G, and gadd 4 5 obtained from the database and the two base sequences inside the restriction enzyme recognition site, the 5 1 2 primers used in this method can be determined In which primer set, that is, the combination of X and Y primers, detected P 2 1, mdm 2, cyclin G, and gadd 4 5. The detected D N A length is also explained. Based on this, among the molecules classified by electrophoresis, peaks corresponding to the corresponding molecular weights are fractionated. Regarding the D N A contained in the divided peaks, the base sequence is determined through the sequence determination. The gene ‘fiP’ p 2 1, m d m 2, c y c 1 i η G, and gadd 4 5 0

The effect of 1-6 radiation on the performance of P 2 1, m d m 2, c y c 1 i n G, and gadd 45. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, the peaks of the genes of P 2 1, m d m 2 and cyclin G obtained according to the above methods are shown in Figs. 9, 10 and 11. The graph in the upper section of each figure is part of an analysis map of m R N A gene expression obtained from unirradiated cells. The lower graph is a part of the analytic map of m R N A from 7 G y irradiated cells over 3 hours. The peaks of the gene of interest are indicated by arrows. As shown in FIGS. 9 to 11, it can be confirmed that the respective expressions of P 2 1, m d m, and c y c 1 i n G can be increased by irradiation of radiation. In addition, although this paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) -28-1237663 A7 B7 V. Description of the invention (26) Although data is not shown, gadd 4 5 also achieved the same result. Example 2 (Please read the notes on the back before filling in this page} The analysis of gene expression frequency and the analysis of gene expression frequency. Cells are divided using yeast (hereinafter, denoted by S · p.) And budding yeast (hereinafter, (Expressed as s.c ·). Extracted in the same manner as in Example 1, and the resulting mixture ratio of total mrn A and the amount of mRNA of S.p. were expressed as 0, 0.02, 0.2, 1, 2, and 2 micrograms. , S · c • The amount of mRNA is 2, 2, 2 '2, 2 and 0, respectively, as shown in Figure 12. For the six m R Ν Α modulators prepared as described above, as in Example Works Gene expression analysis diagrams were prepared as described. Part of the obtained gene expression analysis diagrams are shown in Figure 13. The composition of the mRNA modulators from each diagram is shown on the left. The uppermost diagram shows S · p · genes. A part of the performance analysis chart, and the chart at the bottom is a part of the analysis chart of S _ c · gene performance. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Figure 14 is from S · p · The wave crests are connected by the vertical axis. As shown in Figure 14, the wave The change in the peak size is dependent on the amount of m RNA contained in the m RNA modulator in the m RNA modulator. As can be seen from Examples 1 and 2, a gene expression analysis map was prepared according to the method of the present invention, and the obtained gene expression analysis map was obtained. The gene expression amount shown in the figure fully reflects the amount of m RNA present in the sample. Therefore, the gene expression frequency can be analyzed according to the method of the present invention. According to the method of the present invention, it can be easily made about The paper scale applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) -29- 1237663 A7 B7 V. Description of the invention (27) An analysis map of the gene expression of the expressed genes. In addition, according to the analysis map of such gene expression, then More expression genes can be identified than the previous method, that is, almost all expressed genes can be identified. Moreover, the gene expression analysis map of the fundamental invention aspect can identify each gene for each gene. Furthermore, because, This is to reflect the expression level of a gene, so it is also possible to analyze the expression frequency of a gene. In particular, for unknown genes, the expression frequency can also be analyzed in the same way as a known gene. . Other advantages and changes can be easily conceived by the industry. Therefore, in this broad flanking, the present invention cannot be limited to the detailed description and typical aspects shown here. Therefore, it is not beyond the scope of it. The attached scope of the patent application and the ideological spirit or scope of the overall invention explicitly expressed by the equivalent can be changed in various ways. (Please read the notes on the back before filling out this page.) This paper is printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Standards apply to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) -30- 1237663 A7 B7 V. Description of invention (2S) Sequence Listing < 110〉 AISIN SEIKI KABUSHIKI KAISHA National Institute of Radiological Sciences MAZE INC. Masumi Abe Toshiyuki Sairo < 12ϋ > Method for analysing of gene expression

< 130 > 01S1459P < 150 > JP 2000-377887 < 151〉 2000-12-12 „. Clothes-(Please read the notes on the back before filling out this page) Order the stamp of the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs ≪ 160〉 24 < 170〉 Paieniln version 3. 1 This paper size applies to China National Standard (CNS) A4 (210X297 mm) -31-1237663 A7 B7 V. Description of Invention (29) (Please read the back first Please fill out this page again) < 210 > 1 (211) 22 < 212 > DNA < 213〉 Artificial < 400 > 1! Cgggicgxax cagactigca ca 22 Printed by the Consumers ’Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs < 210 〉 2 < 211 > 20 < 212〉 With < 213 > < 400 > 2 igigcaagtc tgatacgacc 20 This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) -32- 1237663 A7 B7 V. Description of the invention (30) < 210 > 3 < 211 > 22

< 212 > DNA < 213 > artificial 丨 < 400〉 3! I tacatcaggx gtccgatgat re 22 < 21ϋ > 4 < 211) 20 < 212〉 DNA < 213〉 artificial < 400) 4 gaatcatcgg acacctgaig < 210〉 5 This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) · Binding · Order printed by the Intellectual Property Bureau of the Ministry of Economic Affairs Consumer Cooperatives -33 -1237663 A7 B7 V. Description of the invention (31) 丨 Clothing-(Please read the precautions on the back before filling out this page) < 211 > 22 < 212 > DNA (213> Artificial &400; 5cgagtcgtat cagacttgca ca 22 I < 210 > 6 I < 211 > 20 < 212 > DNA < 213〉 Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Artificial Economy < 400 > 6 igigcaagic igaxacgaci: · 2〇 < 210 > 7 copies Paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) -34- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1237663 A7 B7 V. Description of the invention (32) < 211 > 22 < 212 > DNA j < 213 > artificial &400; 7 xacttggact acagtcgtga ca 22 < 210〉 8 〈211〉 20 < 212 > DNA < 213〉 artificial. < 400〉 8 tgtcacgacx gtagiccaag 20 < 210 > 9 < 211 > 116 This paper is in accordance with the Chinese National Standard (CNS) ) A4 specification (210X 297mm) 1 .--- * --------: ---, order ------ «(Please read the notes on the back before filling this page)- 35- 1237663 A7 ____ B7 V. Explanation of the invention (33) < 212 ^ DNA < 213 > Homo sapiens Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs < 400 > 9 gaaaccgggg tgggtggtgt ciccaggica atcaacttct gtacigggct ctgaccacaa 60 gagaggxtx gagggxt cccxccag-ττ aacaaa 116 (210 > 10 < 211 > 116 < 212 > DNA < 213 > Homo sapiens < 400 > 10 gaaaccgggg tgggtggtgt ctccaggtca aicaactict giactgggcx ctgaccacaa 60 tcggtttag iac-txggagxi tacggargia ----- (Please read the precautions on the back before filling this page) The size of the paper used in this edition applies to Chinese National Standard (CNS) A4 (210X297 mm) -36- 1237663 A7 B7 V. Description of the invention 34) < 210 > 11 < 211 > 116

< 212 > DNA < 213 > Homo sapiens < 400 > 11 gaaaccgggg rgggxggigx ciccaggxca atcaactrct gtactgg ^ ct cigaccacaa i tcggttttca gaccacaatg tiaggagggi attixxacai ccciccag----aacaaa-60- I (Please read the notes on the back before filling this page) Order < 210 > 12 < 211 > 116

&lt; 212 &gt; Printed by the Consumer Cooperatives of Intellectual Property Bureau of the Ministry of Economic Affairs &lt; 213 &gt; Homo sapiens &lt; 400〉 12 gaaaccgggg tgggxggtgt ctccaggtca atcaacttct gtactgggct ctgaccacaa 60 This paper is in accordance with the Chinese National Standard (A4) (2l0x297 mm) ) 37- 1237663 A7 B7 V. Description of the invention (35) tcggmtca gaccacaatg txaggagggx atttttacai ccctccagti aacaaa 116 &lt; 210 &gt; 13 &lt; 211 &gt; 116 &lt; 212〉 DNA &lt; 213 &gt; Homo sapiens &lt; 400> 13 ciaggtgtggatgggg gtacigggct ctgaccacaa 60 i I icggxixtca gaccacaatg itaggagggt atttttacai ccctccagti aacaaa 116 &lt; 210 &gt; 14 &lt; 211 &gt; 116 <212> DNA &lt; 213 &gt; Homo sapiens This paper is in accordance with the Chinese National Standard (CNS) 297 A4 standard I ----.---- Clothing-(Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs of the Ministry of Economic Affairs of the People's Republic of China. 1237663 A7 B7 V. Description of the invention (mud) &lt; 400 &gt; 14 gaaaccgggg tgggtggtgi ctccaggrca aicaacttct giacigggct cxgaccacaa icggttitca gaccacaatg itaggagggt aimtacaiaca &lt; cgtcca &lt;116;; 212 &gt; DNA &lt; 213 &gt; Homo sapiens

I! &Lt; 400〉 15 gaaaccgggg igggtggtgt ctccaggtca atcaacttct gxacigggci cxgaccacaa

I icggmica gaccacaatg itaggagggt aitmacai ccctccagit aacaaa

&lt; 210 &gt; 16 &lt; 211 &gt; 116 I &lt; 212〉 DNA &lt; 213 &gt; Homo sapiens This paper size applies the Chinese National Standard (CNS) A4 specification (21〇X 297 mm) (Please read the precautions on the back first Refill this page), r Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs-39- 1237663 A7 B7 V. Invention Description (37) &lt; 400 &gt; 16 gaaaccgggg tgggtggigt ctccaggica axcaacnci gtactgggct ctgaccacaa 60 tcggxittca gaccacaatg ttaggagcc 116 ; 210 &gt; 17 &lt; 211> 116 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens i &lt; 400 &gt; 17 gaaaccgggg tgggiggtgx ciccaggxca atcaacitct gtactgggct ctgaccacaa 60 tcggitxxca gaccacaatg nagazagtgt cc &gt; cc &gt; cc &gt; I --------- (Please read the notes on the back before filling this page)-Γ The paper printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs is compliant with China National Standards (CNS) Α4 specifications (2丨 〇 &gt; &lt; 297 mm) -40-1237663 A7 B7 V. Description of the invention (38)

&lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400〉 18, gaaaccgggg tgggiggtgt ctccaggtca atcaacttcx giacxgggci ctgaccacaa 60 icggttttca gaccacaatg it & gagagtgt aixxtiacat cccicclt &gt; 116 &gt; 210 ·

I i &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 19 gaaaccaggg tgggtggtgi ctccaggtca axcaacrccx gtacigggct ctgaccacaa 60 tcggititca gaccacaatg tiaggagggt aimiacat ccctccagtt aacaaa 116 standard (Standard 210) China 210 Standards (210) China 210 Standards 210 (Please read the precautions on the back before filling out this page)

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs-41-1237663 A7 B7 V. Description of Invention (39) &lt; 211 &gt; 116 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 20 gaaaccgggg rgggtggigi ctccaggtca aicaacttci gxactgggcx cigaccacaa 60 icggtxttca gaccacaatg ttaggagggt aittttacat ccctccagtt aacaaa 116 &lt; 210 &gt; 21

I! '&Lt; 211> 116 I. &Lt; 212 &gt; DNA 〈213〉 Homo sapiens I &lt; 400) 21 gaaaccgggg igggtggigi cxccaggxca atcaacxtci giacigggcx cxgaccacaa 60 tcggttttca gaccacaatg ttaggagggt atttttaiacc cc standard A4 specifications (210X 297 mm) (Please read the notes on the back before filling out this page) Order k9 ·. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs-42- 1237663 A7 B7 V. Description of Invention (4〇) &lt; 210 &gt; 22 &lt; 211 &gt; 116 <212> DNA &lt; 213 &gt; Homo sapiens (Please read the notes on the back before filling out this page) &lt; 400 &gt; 22 gaaaccgggg tgggiggigi ctccaggtca aicaacxtct gtacigggct ctgaccacaa 60! ^ Atggttgca gacc ccctccagtt aacaaa 116! &lt; 210 &gt; 23 &lt; 211 &gt; 116 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 23 This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) Order # Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 43 1237663 A7 B7 V. Description of Invention (41) gaaaccgggg t gggtggigi ctccaggtca atcaacrtci gtacxgggct ctgaccacaa 60 tcggttttca gaccacaatg ttaggagggi aimiacax ccctccagtt aacaaa 116 &lt; 210) 24 &lt; 211> 116 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400, 24 I gaaaccaggg tgggiggtgi ctccaggtca atcaacttci giactgggci ctgaccacaa 60 tcggtitica gaccacaatg ttagga ^ ggt atttxiaiat cccxccagri aacaaa 116 (Please read the notes on the back before filling out this page) _Packing and Ordering Printed by the Intellectual Property Bureau Staff Consumer Cooperatives of the Ministry of Economics This paper is printed in accordance with China National Standard (CNS) A4 (210X297 mm) -44-

Claims (1)

1237663 Printed by A8, B8, C8, D8, Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs ^, _Zhongzhu Special Department i Scope 1 1 · A method for making analysis of gene expression, with the following steps (a) m RNA, a step of synthesizing c DNA to which a target substance is added at its 3 'terminal, (b) a step of cutting the obtained reaction product with a first restriction enzyme X, (c) having a first restriction enzyme The X adaptor in which the sequence of the restriction enzyme cutting site of X is a complementary sequence is a step of combining the fragments obtained in step b. (D) By binding a substance having a high affinity to the target substance, step c can be recovered. In the step of obtaining the fragment, (e) the fragment recovered in step d is cut with a second restriction enzyme Y, and the fragment bound to the target substance is removed, and then a step containing the cut 5 'side fragment of c DNA can be obtained, (f) For the fragment obtained in step e, a step of adding a Y aptamer having a complementary sequence to the sequence of the second restriction enzyme Y recognition cut site, and (g) using a complement to the X aptamer sequence Sex sequence And its primer at the 3 ′ terminal has an arbitrary two base sequence NN, and a primer complementary to the Y adaptor sequence and its primer at the 3 ′ terminal has an arbitrary two base sequence NN. The step of performing a PCR reaction on the fragment, (h) performing the electrophoresis of the obtained PCR product, and preparing the gene expression analysis map by detecting the moving distance and the wave peak. This paper size applies to Chinese National Standard (CNS) A4 specification (ΉΟ × 297mm) (Please read the precautions on the back before filling this page) -45-1237663 A8 B8 C8 D8 VI. Application for Patent Scope Printed by the Consumers' Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 2 Please refer to the Patent Scope Method 5 where the X adaptation subsequence 3 5 terminal has any -Afe: two The base 5 3 terminal addition fluorescent substance is detected by detecting 3 of the fluorescent substance. • If the method of patent scope is requested, the restriction enzyme X and the group are «out; ACCIIASPL EI IB fa I Λ BS h 1 2 3 6 IBS h B s P 1 4 3 I, BS t UC f 〇ICSP 6 ID Η ae III Η a PII Η in 6 I Λ Η i η P 1 I Η s P 9 2 II Η SPA Μ b 〇I Μ S e I Λ Μ s \ N 1 a III Λ Ρ a 1 IS se 9 I Λ Τ aq I Ν T τ ru 9 I Τ s ρ 5 0 9 τ th Η Β 8 I o 4 Method of patent scope, where the restriction enzyme X is M Μ se I 〇 5 • If you ask for the scope of the patent, the paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1 Gene expression analysis diagram of the production column having a complementary sequence and the sequence of the primers N N, and then based thereon Here, the electrophoresis fluorescence amount P C R of the product is carried out. The restriction enzyme Y for the analysis of gene expression in item 1 is the enzymes, AfaI, AluI, B sc FI, I, B si SI, I, B su RI, pnII, FnuDII,, HhaI, Hin2I,, H pa II, I, Kz〇9I, MaeI, pi, MvnI, NdeI I, RsaI, Sau3AI, hal, Trull, I, T sp EI and sp I of the gene expression analysis chart, and the restriction enzyme Y is 1 for the analysis of gene expression (please read the precautions on the back before filling this page), 1T -46-1237663 A8 B8 C8 D8 6. Method of applying for patent scope 3, in which the X adaptor contains The NN included in the NN and Y aptamers is a designer who combines adenine, thymine, guanine, and cytosine, and all 256 kinds of X and Y aptamers are used. 6. The method for making a gene expression analysis map according to item 1 of the scope of patent application, wherein the combination of the target substance and the substance with high affinity to the target substance is selected from the group consisting of biotin and streptavidin, biotin and A group of avidin, FI TC and FI TC antibodies, DIG and anti-DIG, protein A and murine Ig G, and latex particles. 7. The method for making a gene expression analysis chart according to item 1 of the scope of the patent application, which comprises the steps of preparing a gene expression analysis chart after detecting the moving distance and peak reduction by electrophoresis of the PCR product, The peaks recovered are the steps of determining the sequence of the PCR product contained in the sequence by determining the sequence, and identifying the expressed genes. 8 · The method for making a gene expression analysis map according to item 1 of the scope of patent application, which further comprises the identification sequence of the restriction enzyme X and the restriction enzyme Y, and the reaction product obtained in step a to restrict the enzyme X and the restriction enzyme Y. The length of the cut fragments and the data obtained from any database are compared on a computer to identify the expressed genes. 9. A method for making an analysis map of gene expression, comprising the following steps (a): synthesize m DNA extracted from a cell by adding c DNA to a 3 'terminal of the target substance, and divide the obtained synthetic product into two Steps for each fraction, (b) The paper size of the obtained reaction product is cut by the first restriction enzyme X. The paper size of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling in This page) Order: Printed by the Employees 'Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs-47- 1237663 Α8 Β8 C8 D8 Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 6. The scope of patent application 4 steps, (c) will have the first limit Step X of the restriction enzyme of enzyme X is a complementary sequence of the X adaptor to the fragment obtained in step b. (D) The step of recovering the target substance by binding to a substance with a high affinity can be recovered. (c) The fragment obtained in step (c) is cut with the second restriction enzyme Y to remove the fragment bound to the target substance, and then the A step of cutting the 5 'side fragment of c DNA, (f) a step of adding a Y aptamer complementary to the sequence of the second restriction enzyme Y recognition cut site for the fragment obtained in step e, (g ) Use a primer that has a complementary sequence to the X aptamer sequence and 3 and a terminal that has any two base sequences NN, and a primer that has a complementary sequence to the Y aptamer sequence and 3 'terminal that has any two bases A sequence of NN primers, and a step of performing a PCR reaction on the fragment obtained in the foregoing step f, (h) a step of cutting the second synthetic product fraction obtained in step a with the restriction enzyme Y, (i) having Restriction enzyme Y's adaptor at the restriction enzyme cutting site is a complementary sequence of the Y 'aptamer, the step of combining the fragments obtained in step h' (j) by binding a substance with a high affinity to the target substance, Step of recycling the fragments obtained in step i, t paper size is applicable _ National Standard (CNS) A4 size (210X297 mm) (Please read the precautions on the back before filling this page) -48- 1237663 A8 B8 C8 D8 Patent application scope 5 Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs · (k) Cut the fragment recovered in step j with restriction enzyme X to remove the fragment bound to the target substance, Then, a step containing the 5 ′ side fragment of c DNA cleaved can be obtained. (1) For the fragment obtained in step k, an X ′ aptamer having a sequence complementary to the restriction enzyme X recognition cut site is added. (M) using a combination of primers that are complementary to the Y 'aptamer sequence and whose 3' terminal has any two base sequences NN, and complementary to the X 'aptamer sequence A sequence consisting of the primers of the sequence and its 3 ′ terminal having any two base sequences NN, and performing a PCR reaction step, (η) performing electrophoresis on the PCR products obtained in step g and step m, by detecting the moving distance and the peak Steps to make a gene expression analysis map. 10. The method for preparing a gene expression analysis map according to item 9 of the scope of patent application, wherein the X adaptor sequence used in step g has a complementary sequence and its 3 ′ terminal has any two base sequences NN The primer is a fluorescent substance added to its 5 'terminal; and the primer having a complementary sequence to the Y' adaptor sequence used in step m and its 3 'terminal having any two base sequences NN is the A fluorescent substance is added to its 5 'terminal; thereby, the electrophoresis result of the PCR product is performed by detecting the fluorescent amount of the fluorescent substance. 1 1 · If you make a gene expression analysis chart for item 9 of the scope of patent application (please read the notes on the back before filling this page) £ The size of the paper is applicable to the Chinese National Standard (CNS) A4 (210X297 mm)- 49- 1237663 κ, patent application scope ABCD Method printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5 Wherein the limit: enzyme X and the restriction enzyme 〖Υ are selected from the group of enzymes shown in the following [1] ; ACCII &gt; A fa I &gt; A 1 UIASPLEI Λ B fa I, Β S c FI &gt; BS h 1 2 3 6 I Λ BS hi Λ B si SI, BSP 1 4 3 I Β S t UI Λ B su RI, C f 〇ICSP 6 I Dp η II, F nu DII Λ Η ae III Η a PII ^ Η ha I, H in 2 I Η 1 n 6 I Η 1 η P 1 I &gt; Η P a II &gt; P s P 9 2 II Η SPAIK z 〇9 I, Mae I Μ b 0 I, Μ se I Μ sp I, M v η I, N de II, N 1 a III Λ P a II ^ R sa I, S au 3 AIS se 9 I Λ T a QI Λ T ha I ^ Trull τ ru 9 I, T s 5 0 9 1 Λ T s Pl EI and τ th HB 8 I 0 1 2 • The method of making a gene expression analysis chart according to item 9 of the patent, where the restriction enzyme X is MS pi and the restriction enzyme 'F Μ se I 〇1 3 • Method 5 for the analysis of gene expression, as described in item 9 of the three main sundial patents, where the NN contained in the X aptamer and the NN contained in the Y aptamer are glands The designers of the combination of purine Λ thymidine guanine and cytosine all use 2 5 6 kinds of X and Y aptamers. 14. If you ask for the method of making a gene expression analysis chart in item 9 of the patent scope, the combination of the target substance and the substance with high affinity to the target substance is selected from biotin and streptavidin, biological Suhe (Please read the notes on the back before filling this page) This paper size applies to Chinese National Standard (CNS) A4 specification (210 × 297 mm), 1T, -50-1237663 Printed by A8, Consumer Cooperative of Intellectual Property Bureau, Ministry of Economic Affairs B8 C8 D8 6. Scope of patent application 7 Avidin, F ITC and F ITC antibodies, DIG and anti-DIG, protein a and mouse I g G, and latex particles. 15 · According to the method of making a gene expression analysis chart in item 9 of the scope of patent application, after the PCR product is electrophoresed, the moving distance and wave peak are detected, and the step of making a gene expression analysis chart is provided, and then the detection method is provided. The recovered peaks are recovered and the sequence of the PCR products contained in the determined sequence is determined, and the steps of identifying the expressed genes are performed. 16 · According to the method for generating a gene expression analysis map in item 9 of the scope of the patent application, it further comprises the identification sequence of the restriction enzyme X and the restriction enzyme Y, and the reaction product obtained in step a to restrict the enzyme X and the restriction enzyme. The length of the fragment obtained by Y cutting and the data obtained from any database are compared on a computer to identify the expressed genes. 17 · A method for analyzing the frequency of gene expression, including the following steps (1) For both control cells and test cells, the following steps are performed to make a gene expression analysis map; (a) For the sample drawn by the cell The step of synthesizing the m RNA and synthesizing the c DNA of the target substance at its 3 ′ terminal, (b) the step of cutting the obtained reaction product with the first restriction enzyme X, (C) Restriction enzyme X The sequence of the restriction enzyme cutting site is a complementary sequence of the X aptamer, the step of combining the fragments obtained in step b, (d) by binding a substance with a high affinity to the target substance, this paper scale Applicable to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) -51-1237663 A8 B8 C8 D8 6. The scope of patent application 8 can recover the fragment obtained in step c, (e) Cut the fragment recovered in step d with the second restriction enzyme Y to remove the substance bound to the target A fragment containing the c's DNA 5 'side fragment can be obtained. (F) For the fragment obtained in step e', it will have a γ adaptation that is complementary to the sequence of the second restriction enzyme Y recognition of the cut site. (G) Use a primer that has a complementary sequence to the X aptamer sequence and 3 of which has a terminal with any two base sequences NN, and a primer that has a complementary sequence to the Y aptamer sequence and its The 3 ′ terminal has primers of any two base sequences NN, and performs a PCR reaction on the fragment obtained in the foregoing step f. (H) The obtained PCR product is electrophoresed, and the gene expression analysis is made by detecting the moving distance and wave peak. Step of the graph; and (2) the step of analyzing the change of the gene expression frequency of the test cell by comparing the two gene expression analysis maps obtained in the step (1). 1 ·· A method for analyzing the frequency of gene expression, including the following steps (1) For both the control cell and the test cell, the following steps are performed to make a gene expression analysis map; (a) For the sample drawn by the cell Extract the m RNA to synthesize the c DNA of the ^ terminal addition target substance and divide the obtained synthetic product into two fractions. (B) Cut the first synthetic product fraction with the first restriction enzyme X. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the notes on the back before filling this page) — Ordered by the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by the Consumer Cooperatives -52- 1237663 Intellectual Property Bureau of the Ministry of Economic Affairs A8, B8, C8, D8 printed by the employee's consumer cooperative. 6. In step 9 of applying for a patent, (C) an X aptamer that has a complementary sequence to the restriction enzyme cutting site of the first restriction enzyme X. For step b, The step of combining the fragments, (d) By combining a substance with a high affinity for the target substance, the step of the fragment obtained in step c can be recovered, and (e) the step d is recovered. The fragment is cut with the second restriction enzyme Y, and the fragment bound to the target substance is removed, and a step containing the c '5' side fragment of cleaved DNA can be obtained. (F) For the fragment obtained in step e, the The step of adding the Y adaptor whose complementary sequence of the two restriction enzyme Y recognition cut-off site is complementary, (g) using a complementary sequence to the X adaptor sequence and 3, the terminal having any two base sequences A primer of NN and a primer of complementarity to the Y adaptor sequence and a primer of any two base sequence NN at its 3 ′ terminal, and a step of performing a PCR reaction on the fragment obtained in the foregoing step (h) The second synthetic product fraction obtained by a step of cleaving with restriction enzyme γ, (i) a Y ′ aptamer having a complementary sequence to the restriction enzyme cleaving site of the restriction enzyme Υ, for step h The step of combining the fragments, (j) The step of recovering the fragment obtained in step i by combining the substance with high affinity to the target substance 'This paper standard applies to China National Standards (CNS) A4 Grid (210X297 mm) (Please read the back of the precautions to fill out this page) -53- 1237663 A8 B8 C8 D8 VI. Patent application scope 10 (k) Cut the fragment recovered in step j with restriction enzyme X, and remove the fragment bound to the target substance, then you can get the c Step of DNA 5 'side fragment, (1) For the fragment obtained in step k', a step of adding an X 'aptamer having a complementary sequence to the sequence of the restriction enzyme X recognition cut site, (m) using A combination of primers having complements to the Y 'adaptor sequence and 3' terminal having any two base sequences NN 'and having complements to the X' adaptor sequence and 3 'terminal having any two A combination of primers of a base sequence NN to perform a PCR reaction, (η) a step of electrophoresis of the PCR product obtained in step g and step m, and a step of generating an analysis map of gene expression by detecting a moving distance and a peak; And (2) the step of analyzing the change in the gene expression frequency of the test cell by comparing the two gene expression analysis maps obtained in step 1. (Please read the precautions on the back before filling out this page). Binding and printing Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs This paper applies the Chinese National Standard (CNS) A4 specification (21 ×: 297 mm)